Anti erα sc 543

To measure the effect of E2, BPA and melatonin treatment on OCT4 and ER protein expression, 5 × 10⁵ MCF-7 mammospheres were grown in cell suspension and treated with 10 μM BPA and 10 nM E2 with or without 1 mM melatonin for 6 days. After 6 days, mammospheres were harvested and the pellet was snap- frozen in liquid nitrogen. Cell extracts were thawed in lysis buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100) and centrifuged. The supernatant was used in Bradford assays and Western blot assays. Equal amounts of whole cell extracts (50 μg) were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes for Western analyses. Endogenous proteins were detected by using the following antibodies: anti- OCT4 (ab27985, mouse polyclonal, 1: 1 000, Abcam), anti-ERα (SC-543, mouse polyclonal, 1: 1 000 SantaCruz Biotechnologies) and anti-β-actin (sc-47778, mouse monoclonal, 1: 5 000 SantaCruz Biotechnologies). The antibody incubation was performed in 5% milk in TBST (20 mM Tris HCl, pH 7.5, 120 mM NaCl, 0.1% Tween20). Blots were developed using peroxidase-conjugated goat anti-mouse secondary antibodies as appropriate (1: 5 000, ThermoScientific, Waltham, MA) and SuperSignal WestPico Chemiluminescent substrate (ThermoScientific). Quantification was performed using Image J software as image analyzer.